Identification of Sperm Factor in Human Sperm.

该研究项目的目的是分析精子因子被转移到卵子中并激活卵子。关于卵的激活机制还有许多未阐明的问题。我们假设磷脂酶C（PLC）zeta（PLCζ）是真正的精子因子，并进行了以下研究：（1）用人PLCζ cDNA探针筛选人cDNA文库。(2)We克隆了两个不同的cDNA一个克隆对应于全长PLC cDNA（野生型PLC cDNA），另一个cDNA克隆缺乏外显子2 '-3-4（del. 2 '-3-4变体PLC cDNA）。(3)To对野生型PLC cDNA和del. 2 '-3-4变异型PLC β cDNA，我们合成并纯化了野生型PLC β cDNA和缺失型PLC β cDNA。2 '-3-4变体PLC cDNA使用原核GST融合蛋白表达系统。(4)We将重组蛋白显微注射到小鼠卵中，并使用共聚焦激光显微镜分析细胞内钙离子水平的变化。(5)As结果，显微注射野生型PLC β和del，在小鼠卵中诱发Ca振荡。2 '-3-4变异体PLC β 1可能是一种真正的精子因子，本研究合成的重组PLC β 1可能对治疗ICSI不能激活卵子的不育症有重要意义。

The aim of the research project is analysis of the sperm factor is transferred into in an egg and elicits activation of an egg. There are many unelucidated issues about the mechanism of the egg activation. We hypothesized that phospholipase C(PLC) zeta (PLCζ) which is a sperm isoform of PLC is the true sperm factor, and carried out the following studies;(1)We screened the human cDNA library by the human PLCζ cDNA probe.(2)We cloned two distinct cDNAs. One clone corresponed to the full length PLCζ cDNA (wild type PLCζ cDNA, and another cDNA clone lacked exon 2'-3-4 (del. 2'-3-4 variant PLCζ cDNA).(3)To analyze of the function of the protein encoded by the wild type PLCζ cDNA and del. 2'-3-4 variant PLCζ cDNA, we synthesized and purified the wild type PLCζ and the del. 2'-3-4 variant PLCζ using prokaryotic GST-fusion protein expression system.(4)We microinjected the recombinant proteins into a mouse egg and analyzed changes in intracellular calcium ion level using confocal laser microscope.(5)As a result, ca oscillation was evoked in a mouse egg by microinjection of wild type PLCζ as well as del. 2'-3-4 variant PLCζ, indicating that PLCζ may be a true sperm factor.The recombinat PLCζ synthesized in the present study might be very useful for the treatment of a certain infertility male whose sperm can not activate the egg by ICSI.